key: cord-0921792-d9zlvvq8 authors: Chen, Xuesong; Geiger, Jonathan D. title: Janus sword actions of chloroquine and hydroxychloroquine against COVID-19 date: 2020-07-03 journal: Cell Signal DOI: 10.1016/j.cellsig.2020.109706 sha: c9bd575e17d5112f42429f351942db1c38aedf1b doc_id: 921792 cord_uid: d9zlvvq8 Chloroquine (CQ) and its analogue hydroxychloroquine (HCQ) have been thrust into our everyday vernacular because some believe, based on very limited basic and clinical data, that they might be helpful in preventing and/or lessening the severity of the pandemic coronavirus disease 2019 (COVID-19). However, lacking is a temperance in enthusiasm for their possible use as well as sufficient perspective on their effects and side-effects. CQ and HCQ have well-known properties of being diprotic weak bases that preferentially accumulate in acidic organelles (endolysosomes and Golgi apparatus) and neutralize luminal pH of acidic organelles. These primary actions of CQ and HCQ are responsible for their anti-malarial effects; malaria parasites rely on acidic digestive vacuoles for survival. Similarly, de-acidification of endolysosomes and Golgi by CQ and HCQ may block severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) integration into host cells because SARS-CoV-2 may require an acidic environment for its entry and for its ability to bud and infect bystander cells. Further, de-acidification of endolysosomes and Golgi may underly the immunosuppressive effects of these two drugs. However, modern cell biology studies have shown clearly that de-acidification results in profound changes in the structure, function and cellular positioning of endolysosomes and Golgi, in signaling between these organelles and other subcellular organelles, and in fundamental cellular functions. Thus, studying the possible therapeutic effects of CQ and HCQ against COVID-19 must occur concurrent with studies of the extent to which these drugs affect organellar and cell biology. When comprehensively examined, a better understanding of the Janus sword actions of these and other drugs might yield better decisions and better outcomes. The endolysosome system is a dynamic interconnected network with morphological and functional heterogeneity, and this system exhibits complex interactions with other organelles. Extracellular macromolecules and membrane components are up-taken into endosomes by a variety of endocytic pathways and can either be trafficked through early endosomes to recycling endosomes, which mediates receptor recycling to plasma membrane or Golgi apparatus, or can transition to late endosomes and fusion with lysosomes [26, [31] [32] [33] . As mentioned above, a hallmark feature of the endolysosome system is their acidic luminal pH [25] [26] [27] [28] that is critical for the activity of up to 60 different pH sensitive hydrolytic enzymes including proteases, lipases and nucleases [33] . Early endosomes act as a major sorting station and their acidic nature enables internalized ligands to be dissociated from the endocytosed cell surface membrane receptors to which they were bound. The receptors unbound with ligand can then be recycled back to the cell surface or can traffic to Golgi apparatus via recycling endosomes; dissociated ligands are transported through late endosomes to lysosomes for degradation. The maturation of early endosomes to late endosomes is characterized by an increased number of intralumenal vesicles and the formation of multivesicular through the multidrug resistance protein p-glycoprotein [97] [98] [99] .Through their ability to deacidify acidic organelles along endocytic and biosynthetic secretory pathways, CQ and HCQ disturb many key aspects of cell biology including organellar biology and interorganellar signaling, many of which are linked to their antiviral and immunosuppressive effects. Decades of clinical usage have shown that CQ and HCQ are relatively safe drugs. They are even commonly used during pregnancy in patients with autoimmune disorders. Recent systematic reviews and meta-analyses suggest that maternal HCQ use during pregnancy does not increase risk of major congenital malformations [100, 101] . The reported tissue side effects of CQ and HCQ such as retinopathy [102, 103] , cardiomyopathy [104] , neuromyotoxicity [105] are likely due to abnormal accumulation of these drugs in tissues with chronic use [12, 106] . The unexpected high rate of side effects including cardiac arrhythmias during the COVID-19 pandemic may be related to higher doses of HCQ used, the older ages of the patients, and to drug-drug interactions Other enveloped viruses like influenza A and Ebola, also use the acidic environment of endosomes or endolysosome hydrolases to drive the fusion of viral membranes with endosome membranes and the release of viral genomic content into the cytoplasm [121] . As such, endolysosome de-acidification with a v-ATPase inhibitor [122] or CQ [123, 124] has been used frequently to inhibit cellular entry of enveloped virus in vitro. In the context of COVID-19, CQ and HCQ are often used in combination with antivirals and other drugs such as azithromycin and zinc [130, 131] . By de-acidifying acidic organelles, CQ and CQ could alter the cellular distribution of these drugs. Azithromycin, a macrolide antibiotic, has been used against Zika [132] and Ebola viruses [133] . In vitro evidence indicates that HCQ and azithromycin show synergistic effects on inhibiting SARS-CoV-2 [130] . As a weak base, azithromycin (pKa = 8.5) also tends to accumulate in acidic environments [134] . By de-acidifying acidic organelles, CQ and HCQ could prevent the accumulation of azithromycin in acidic organelles and increase its concentrations in cytosol where azithromycin exerts inhibitory effects on viral replication [130] . On the other hand, such CQ-and HCQ-induced cellular redistributions of azithromycin may exaggerate its QT prolongation effects [135] as observed in the context of COVID-19 [136] . In vitro evidence indicates that zinc inhibits SARS-CoV [137] . In a preprint paper [131] , the authors reason that CQ, as a zinc ionophore [138] , could increase cytosolic concentrations of zinc to inhibit RNA-dependent RNA polymerase. This is contrary to the original observation that CQ is a zinc ionophore [138] , in which chloroquine was observed as a zinc ionophore that targets zinc to lysosomes. Nonetheless, zinc has been shown to inhibit the activity of furin [139] and cathepsins [140] . Interestingly, the activity of furin is also pH-dependent; furin's optimum pH is 6.0 [141] . Thus, in addition to its de-acidifying effects, CQ-induced accumulation of zinc in J o u r n a l P r e -p r o o f endolysosomes could further inhibit activities of furin and cathepsins that are responsible for cleavage of SARS-CoV spike proteins and viral entry. De-acidifying acidic organelles may not be the only mechanism whereby CQ and HCQ interfere with the biology of SARS-CoV2. In human epithelial lung cells, CQ inhibits the phosphorylation of p38 mitogen-activated protein kinase (MAPK) and inhibits the release of human coronavirus from infected cells [142] . Thus, CQ and HCQ may exert its effects on SARS-CoV-2 via altering protein kinase activities. Concerns about the possible antiviral effects of CQ and HCQ: Currently, there exist very limited evidence supporting the use of CQ and HCQ against SARS-CoV-2 and COVID- 19 . First, almost all in vitro studies reported to date were conducted in epithelioid cells derived from the kidney of African green monkeys (Vero E6 cells) or human hepatomas (Huh7 cells) [129, 143, 144] . Vero E6 cells are extremely permissiveness for viral replication including coronaviruses, which is due in part to genetic defects in interferon production [145, 146] and defects in innate antiviral responses [147] . Huh7 cells are also highly permissive for virus replication due to defective retinoic acid-inducible gene I signaling, impaired interferon signaling, and defects in innate antiviral responses [148, 149] . Thus, it is not clear how finding from these cells are translatable to human clinical trials. Second, CQ and HCQ change the pH of endolysosomes and Golgi and cause multiple morphological and functional changes to these organelles including swelling and altered exocytotic release of virus. These changes may result in the formation of intracellular SARS-CoV-2 reservoirs capable of being re-activated when pH normalizes. Most recently (June 23, 2020), several clinical studies have reported on the favorable use of CQ and HCQ for the treatment of COVID-19 [150] [151] [152] [153] [154] [155] [156] [157] [158] [159] . However, other studies have reported no significant beneficial effects and some detrimental effects [160] [161] [162] . Multiple clinical trials testing the possible effectiveness of CQ and HCQ against COVID-19 are ongoing [163] , the results of these studies will be important as more people require treatments for COVID-19. The immunomodulatory properties of CQ and HCQ have long been recognized and these drugs continue to be used clinically for the treatment of rheumatoid arthritis, systemic lupus erythematosus and other inflammatory rheumatic diseases [164] . Although their mechanisms of action remain under investigation, the immunomodulatory effects are likely due to their accumulation in and de-acidification of acidic compartments; endolysosomes and Golgi apparatus of immune cells. The importance of CQ-and HCQ-induced de-acidification as an important mediator in immune modulation may be due to impaired maturation of lysosomes and blockade of fusions between autophagosomes and lysosomes. Indeed, lysosomal degradation of endocytosed or autophagocytosed proteins affects antigen processing and MHC class II presentation [165, 166] . This is consistent with findings that CQ and HCQ inhibit MHC class II expression, antigen presentation and immune activation [167] . Further, RNA and DNA binding to toll-like receptor 7 (TLR7) and TLR9 in endosomes results in TLR signaling activation and production of pro-inflammatory cytokines [168] . Thus, CQ-and HCQ- Membrane bound vesicles in the endocytic pathway (early endosome, recycling endosome, late endosome, and lysosomes) and the biosynthetic secretory pathway (Golgi apparatus and secretory vesicles) all display varying degrees of acidity, and these vesicles rapidly acidify as they progress along the endocytic or secretory pathway. As diprotic weak bases, CQ and HCQ are taken up by cells and trapped in these acidic organelles, where they neutralize pH and alter their structure, function, and trafficking. Hydroxychloroquine-related retinal toxicity The blood-retinal barrier in chloroquine retinopathy Chloroquine transport via the malaria parasite's chloroquine resistance transporter Intracellular pH Plasmodium falciparum neutral aminopeptidases: new targets for antimalarials Antimalarial agents: mechanisms of action Quantitative pH measurements in Plasmodium falciparum-infected erythrocytes using pHluorin Inhibition of the peroxidative degradation of haem as the basis of action of chloroquine and other quinoline antimalarials The pH of the secretory pathway: measurement, determinants, and regulation pH Homeostasis of cellular organelles Lysosomal acidification mechanisms Endosome maturation Regulation of V-ATPase activity Ion and Redox Homeostasis: How Much Do They Really Matter? Organelle acidification and disease Endocytic delivery to lysosomes mediated by concurrent fusion and kissing events in living cells Lysosomes: fusion and function The lysosome turns fifty Role of LBPA and Alix in multivesicular liposome formation and endosome organization Tor comes to the fore in autophagy Historical landmarks of autophagy research Mechanisms and functions of lysosome positioning Sensors and regulators of intracellular pH pH-dependent regulation of lysosomal calcium in macrophages Cytoplasmic vacuolation of mouse peritoneal macrophages and the uptake into lysosomes of weakly basic substances Antiretroviral Drugs Promote Amyloidogenesis by De-Acidifying Endolysosomes Inhibitors of V-ATPases: old and new players Acidifying Endolysosomes Prevented Low-Density Lipoprotein-Induced Amyloidogenesis Role of endolysosomes in HIV-1 Tatinduced neurotoxicity HIV-1 gp120 Promotes Lysosomal Exocytosis in Human Schwann Cells Modulation of macrophage lysosomal pH by Mycobacterium tuberculosis-derived proteins Regulation of vacuolar pH and its modulation by some microbial species A TRP Channel Senses Lysosome Neutralization by Pathogens to Trigger Their Expulsion Coxiella burnetii Type 4B Secretion System-dependent manipulation of endolysosomal maturation is required for bacterial growth Disorders of lysosomal acidification-The emerging role of v-ATPase in aging and neurodegenerative disease Two-pore channels regulate Tat endolysosome escape and Tatmediated HIV-1 LTR transactivation BK channels regulate extracellular Tat-mediated HIV-1 LTR transactivation Lysosomal positioning coordinates cellular nutrient responses The lysosome as a cellular centre for signalling, metabolism and quality control Soluble adenylyl cyclase is essential for proper lysosomal acidification Molecular mechanisms of cutis laxa-and distal renal tubular acidosis-causing mutations in V-ATPase a subunits, ATP6V0A2 and ATP6V0A4 Chloride and the endosomal-lysosomal pathway: emerging roles of CLC chloride transporters An inside job: how endosomal Na(+)/H(+) exchangers link to autism and neurological disease Potential role for white matter lysosome expansion in HIV-associated dementia Autophagy: an overlooked mechanism of HIV-1 pathogenesis and neuroAIDS? Human immunodeficiency virus type-1 infection inhibits autophagy APOE epsilon4 moderates abnormal CSF-abeta-42 levels, while neurocognitive impairment is associated with abnormal CSF tau levels in HIV+ individuals -a cross-sectional observational study Chloroquine and its analogs: a new promise of an old drug for effective and safe cancer therapies Massive cell vacuolization induced by organic amines such as procainamide Cation trapping by cellular acidic compartments: beyond the concept of lysosomotropic drugs Induction of lysosomal dilatation, arrested autophagy, and cell death by chloroquine in cultured ARPE-19 cells Chloroquine inhibits autophagic flux by decreasing autophagosome-lysosome fusion The position of lysosomes within the cell determines their luminal pH Modulating lysosomal function through lysosome membrane permeabilization or autophagy suppression restores sensitivity to cisplatin in refractory non-small-cell lung cancer cells Lysosome Membrane Permeabilization and Disruption of the Molecular Target of Rapamycin (mTOR)-Lysosome Interaction Are Associated with the Inhibition of Lung Cancer Cell Proliferation by a Chloroquinoline Analog Chloroquine inhibits intracellular degradation but not secretion of Alzheimer beta/A4 amyloid precursor protein Lysosomal function and dysfunction: mechanism and disease Lysosomal membrane permeabilization and cell death Mitochondrial membrane permeabilization is a critical step of lysosome-initiated apoptosis induced by hydroxychloroquine Defective acidification of intracellular organelles results in aberrant secretion of cathepsin D in cancer cells Interfering with endolysosomal trafficking enhances release of bioactive exosomes Neutralization of pH in the Golgi apparatus causes redistribution of glycosyltransferases and changes in the O-glycosylation of mucins Elevated Golgi pH in breast and colorectal cancer cells correlates with the expression of oncofetal carbohydrate T-antigen Milieu-induced, selective aggregation of regulated secretory proteins in the trans-Golgi network Retrograde transport from the pre-Golgi intermediate compartment and the Golgi complex is affected by the vacuolar H+-ATPase inhibitor bafilomycin A1 Mannose 6-phosphate receptors: new twists in the tale Sequence and overexpression of GPP130/GIMPc: evidence for saturable pH-sensitive targeting of a type II early Golgi membrane protein Cycling of early Golgi proteins via the cell surface and endosomes upon lumenal pH disruption Ionic milieu controls the compartment-specific activation of pro-opiomelanocortin processing in AtT-20 cells Impaired glycosylation and cutis laxa caused by mutations in the vesicular H+-ATPase subunit ATP6V0A2 A recurrent missense variant in SLC9A7 causes nonsyndromic X-linked intellectual disability with alteration of Golgi acidification and aberrant glycosylation Chloroquine and ammonium chloride prevent terminal glycosylation of immunoglobulins in plasma cells without affecting secretion Primaquine blocks transport by inhibiting the formation of functional transport vesicles Weakly basic amines inhibit the proteolytic conversion of proalbumin to serum albumin in cultured rat hepatocytes Redistribution of mannose-6-phosphate receptors induced by tunicamycin and chloroquine Prohormone processing in the trans-Golgi network: endoproteolytic cleavage of prosomatostatin and formation of nascent secretory vesicles in permeabilized cells Chloroquine diverts ACTH from a regulated to a constitutive secretory pathway in AtT-20 cells Conversion of proinsulin to insulin occurs coordinately with acidification of maturing secretory vesicles Direct binding of chloroquine to the multidrug resistance protein (MRP): possible role for MRP in chloroquine drug transport and resistance in tumor cells Effects of chloroquine on viral infections: an old drug against today's diseases? Lysosomotropic drugs activate TFEB via lysosomal membrane fluidization and consequent inhibition of mTORC1 activity Systematic review of hydroxychloroquine use in pregnant patients with autoimmune diseases Reproductive outcomes following hydroxychloroquine use for autoimmune diseases: a systematic review and metaanalysis The Ocular Deposition of Chloroquine Chloroquine and hydroxychloroquine binding to melanin: Some possible consequences for pathologies Diagnosis of chloroquine cardiomyopathy by endomyocardial biopsy Chloroquine neuromyotoxicity. Clinical and pathologic perspective Tissue distribution of chloroquine, hydroxychloroquine, and desethylchloroquine in the rat COVID-19 infection: Origin, transmission, and characteristics of human coronaviruses Viral escape from endosomes and host detection at a glance Receptor Recognition by the Novel Coronavirus from Wuhan: an Analysis Based on Decade-Long Structural Studies of SARS Coronavirus Substantial undocumented infection facilitates the rapid dissemination of novel coronavirus (SARS-CoV2) Evidence that TMPRSS2 activates the severe acute respiratory syndrome coronavirus spike protein for membrane fusion and reduces viral control by the humoral immune response Inhibitors of cathepsin L prevent severe acute respiratory syndrome coronavirus entry Characterization of severe acute respiratory syndrome-associated coronavirus (SARS-CoV) spike glycoprotein-mediated viral entry Nabel, pH-dependent entry of severe acute respiratory syndrome coronavirus is mediated by the spike glycoprotein and enhanced by dendritic cell transfer through DC-SIGN SARS coronavirus entry into host cells through a novel clathrin-and caveolae-independent endocytic pathway Cathepsin L functionally cleaves the severe acute respiratory syndrome coronavirus class I fusion protein upstream of rather than adjacent to the fusion peptide A Multibasic Cleavage Site in the Spike Protein of SARS-CoV-2 Is Essential for Infection of Human Lung Cells Proliferative growth of SARS coronavirus in Vero E6 cells Fusion of Enveloped Viruses in Endosomes Inhibitory effect of bafilomycin A1, a specific inhibitor of vacuolar-type proton pump, on the growth of influenza A and B viruses in MDCK cells In vitro inhibition of human influenza A virus replication by chloroquine Different pH requirements are associated with divergent inhibitory effects of chloroquine on human and avian influenza A viruses Characterization of herpes simplex viruscontaining organelles by subcellular fractionation: role for organelle acidification in assembly of infectious particles Anti-HIV effects of chloroquine: inhibition of viral particle glycosylation and synergism with protease inhibitors Acidotropic amines inhibit proteolytic processing of flavivirus prM protein The Use of Antimalarial Drugs against Viral Infection New insights on the antiviral effects of chloroquine against coronavirus: what to expect for COVID-19? In vitro testing of combined hydroxychloroquine and azithromycin on SARS-CoV-2 shows synergistic effect Hydroxychloroquine and azithromycin plus zinc vs hydroxychloroquine and azithromycin alone: outcomes in hospitalized COVID-19 patients Zika virus cell tropism in the developing human brain and inhibition by azithromycin Evaluation of Ebola Virus Inhibitors for Drug Repurposing Pharmacokinetics of a single 1g dose of azithromycin in rectal tissue in men The QT interval in patients with COVID-19 treated with hydroxychloroquine and azithromycin Zn(2+) inhibits coronavirus and arterivirus RNA polymerase activity in vitro and zinc ionophores block the replication of these viruses in cell culture Chloroquine is a zinc ionophore Furin inhibition by compounds of copper and zinc Lysosomal metal, redox and proton cycles influencing the CysHis cathepsin reaction Activation of the furin endoprotease is a multiple-step process: requirements for acidification and internal propeptide cleavage Inhibition of human coronavirus 229E infection in human epithelial lung cells (L132) by chloroquine: involvement of p38 MAPK and ERK Chloroquine and hydroxychloroquine as available weapons to fight COVID-19 Chloroquine for the 2019 novel coronavirus SARS-CoV-2 Regulation of the interferon system: evidence that Vero cells have a genetic defect in interferon production Transcriptional and posttranscriptional regulation of exogenous human beta interferon gene in simian cells defective in interferon synthesis Characterization of the interferon regulatory factor 3-mediated antiviral response in a cell line deficient for IFN production Regulating intracellular antiviral defense and permissiveness to hepatitis C virus RNA replication through a cellular RNA helicase, RIG-I Innate immune responses in human hepatocyte-derived cell lines alter genotype 1 hepatitis E virus replication efficiencies Hydroxychloroquine and azithromycin as a treatment of COVID-19: results of an open-label non-randomized clinical trial Azithromycin and Hydroxychloroquine Accelerate Recovery of Outpatients with Mild/Moderate COVID-19 Empirical treatment with hydroxychloroquine and azithromycin for suspected cases of COVID-19 followed-up by telemedicine Hydroxychloroquine plus J o u r n a l P r e -p r o o f Journal Pre-proof azithromycin: a potential interest in reducing in-hospital morbidity due to COVID-19 pneumonia Treatment Response to Hydroxychloroquine, Lopinavir/Ritonavir, and Antibiotics for Moderate COVID 19: A First Report on the Pharmacological Outcomes from South Korea Negative nasopharyngeal SARS-CoV-2 PCR conversion in Response to different therapeutic interventions Efficacy of hydroxychloroquine in patients with COVID-19: results of a randomized clinical trial Treating COVID-19 with Chloroquine Hydroxychloroquine in patients mainly with mild to moderate COVID-19: an open-label, randomized Outcomes of hydroxychloroquine usage in United States veterans hospitalized with Covid-19 Outcomes of Hydroxychloroquine Treatment Among Hospitalized COVID-19 Patients in the United States-Real-World Evidence From a Federated Electronic Medical Record Network Association of Treatment With Hydroxychloroquine or Azithromycin With In-Hospital Mortality in Patients With COVID-19 in A systematic review on the efficacy and safety of chloroquine for the treatment of COVID-19 Mechanisms of action of hydroxychloroquine and chloroquine: implications for rheumatology Intracellular transport of class II MHC molecules directed by invariant chain Autophagy Beyond Intracellular MHC Class II Antigen Presentation Targeting proteins to distinct subcellular compartments reveals unique requirements for MHC class I and II presentation The ectodomain of Toll-like receptor 9 is cleaved to generate a functional receptor Mechanism of endosomal TLR inhibition by antimalarial drugs and imidazoquinolines Chloroquine inhibits production of TNF-alpha, IL-1beta and IL-6 from lipopolysaccharide-stimulated human monocytes/macrophages by different modes Chloroquine inhibits processing of tumor necrosis factor in lipopolysaccharide-stimulated RAW 264.7 macrophages Cytokine Secretion in Macrophages: SNAREs, Rabs, and Membrane Trafficking Translocation of interleukin-1beta into a vesicle intermediate in autophagy-mediated secretion Different Members of the IL-1 Family Come Out in Different Ways: DAMPs vs Elevated levels of IL-6 and CRP predict the need for mechanical ventilation in COVID-19 Chloroquine decreases cell-surface expression of tumour necrosis factor receptors in human histiocytic U-937 cells HCQ could reduce the secretion of, for example, the proinflammatory cytokine TNFα, IL-1β and IL-6